Human Α-globin Research Articles

Lnp-Targeting Hematopoietic Stem Cells and Lentiviral Gene Transfer to Generate and Rescue a Novel Mouse Model of Lethal Alpha-Thalassemia

α-Thalassemia (α-thal) is caused by insufficient production of the α-globin protein. Clinical presentation of α-thal varies from an asymptomatic condition (one inactivated α-globin gene) to Bart's Hydrops Fetalis Syndrome/BHFS (complete knockout). In patients with severe α-thal, allogeneic bone marrow transplantation may be required for survival. Alternatively, patients could be treated through autologous BMT following ex vivo transduction with a human α-globin expressing lentiviral vector. However, a proper model system and vector must be established for the development of such a treatment. We crossed animals that had deletion of both Hba-a1 and Hba-a2 in heterozygosity (Hba-a1+/ko/Hba-a2+/ko) to generate knockout (KO) embryonic mice lacking all the α-globin genes (Hba-a1ko/ko/Hba-a2ko/ko). Although these embryos die perinatally, their fetal liver cells (FLC) at 13.5 dpc are still viable and represent a source of engraftable hematopoietic stem cells (HSC). KO-FLC were transplanted into congenic myeloablated WT mice to generate mice carrying Hba-a1ko/ko/Hba-a2ko/ko HSC. These animals developed a severe phenotype ~7 weeks post engraftment. As these mice lack α-globin expression, we observed β-tetramer formation (β4, HbH, Fig1A), resulting in aberrant red blood cell (RBC) morphology, β-globin precipitates, high RBC oxygen binding affinity, elevated erythropoietin, hematocrit and hemoglobin levels, splenomegaly and vaso-occlusive episodes, due to the large number of abnormal RBCs. Furthermore, we observed iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver. However, FLC engraftment is cumbersome and requires extensive breeding and many animals to generate enough FLC for few recipients. To facilitate the generation of α-thal mice, we created conditional knockout (cKO) mice that have been edited to remove Hba-a2, while inserting loxP sites flanking Hba-a1 (Hba-a1fl/Hba-a2ko). Homozygous cKO animals (Hba-a1fl/fl/Hba-a2ko/ko) demonstrate a mild phenotype. To achieve complete deletion of the α-globin genes (Hba-a1ko/ko/Hba-a2ko/ko) in HSC, we used lipid nanoparticles (LNPs), which are one of the most promising delivery systems to package and make mRNA into a useful therapeutic. We developed a novel LNP preparation with an antibody to target CD117 and deliver nucleoside-modified mRNA to HSC, (see abstract Breda et al). We treated HSC from homozygous cKO animals (Hba-a1fl/fl/Hba-a2ko/ko) ex vivo with CD117-LNP embedded with Cre mRNAs (Cre-CD117-LNP). Cells showed the expected deletion of the α-globin genes and were injected into myeloablated recipient mice. These chimeras recapitulated the phenotype observed following transplantation of constitutive α-globin-KO FLC, including elevated hematocrit, splenomegaly, and culminating in lethality ~7 weeks post-transplant. We screened multiple potential erythroid specific lentiviral vectors in both mouse and human cell lines and identified a promising candidate, ALS20αI, which at VCN=1 produced human α-globin protein equivalent to that of a single endogenous α-globin gene. Myeloablated recipient mice transplanted with Cre-CD117-LNP-treated cKO BM die roughly 7 weeks post-transplant with the expected pathological phenotype (Fig1A). In contrast, mice receiving BM treated with Cre-CD117-LNP and ALS20αI (VCN>1) all survived long term (>4-month) with normalization of erythropoiesis and iron metabolism. VCNs were proportional to chimeric hemoglobin (human α/mouse β) levels and concurrently decreased or absent HbH (Fig1B-C-D). Additionally, we tested ALS20αI in erythroid progenitors derived from CD34 cells isolated from patients with deletional and non-deletional HbH disease. Preliminary data demonstrates improvement in α-globin/β-globin mRNA ratio and reduction in formation of HbH by HPLC. In summary, we have developed new mouse models for α-thal with a lethal and reproducible phenotype. Remarkably, use of LNP technology accelerated the generation of α-thal mice and their characterization. Furthermore, our new vector ALS20αI rescued these animals with stable expression of the human α-globin gene. These data strongly support the use of ALS20αI for the cure of severe forms of α-thal, as 2 copies may be sufficient to rescue patients affected by BHFS, while 1 copy may be sufficient to cure patients affected by HbH disease. HP, SR corresponding Authors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Severe Mouse Model of Alpha-Thalassemia to Study Abnormal Iron Metabolism and Erythropoiesis, Hematopoietic Stem Cell Behavior and Development of a Gene Therapy Approach for Its Treatment

Alpha thalassemia (α-thal) is caused by insufficient production of the α-globin protein because of either deletional or non-deletional inactivation of endogenous α-globin genes. Clinical presentation of deletional α-thal varies from an asymptomatic condition (one inactivated α-globin gene) to a complete knockout (Hb Bart's Hydrops Fetalis). In patients with severe α-thal, a blood transfusion independent state is achievable through allogeneic bone marrow transplantation.The aims of this study are to develop a novel adult mouse model of α-thal and a gene therapy approach for this disease.We generated adult animals that do not produce α-globin chains (α-KO) through transplantation of homozygous B6.129S7-Hbatm1Paz/J fetal liver cells (FLC; isolated at E14.5) into WT recipient mice. These animals demonstrate a worsening phenotype, paradoxically showing elevated hematocrit, high reticulocyte count and a high number of red blood cells (RBC) which expressed only β-globin chains (HbH). RBC show aberrant morphology and aggregation of α- globin tetramers on RBC membranes. Due to severe inability of these RBC to deliver oxygen, the mice eventually succumb to anemia, showing splenomegaly and other organ pathologies, including vaso-occlusive events. These animals show iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver, and elevated erythropoietin (EPO) in the kidney.Interestingly, α-KO embryos show lower numbers of FLC compared to WT embryos, lower frequency of engraftable hematopoietic stem cells (HSC; Lin-Sca-1+c-kit+CD48-), decreased clonogenic potential (fewer class 4 CFUs) and elevated erythroferrone. Lethally irradiated mice transplanted with FLC-KO require 5-6x as many cells as those transplanted with FLC-WT for recovery, further suggesting some level of engraftment impairment. Our current hypothesis is that excessive hypoxia in the embryos impairs HSC function and stem cell fitness. Additional assays are in progress to assess the nature of this impairment.To generate a gene therapy tool to rescue these animals and eventually cure severe human α-thal patients, we screened multiple lentiviral vectors to identify the variant capable of producing the highest human α-globin protein per copy. The selection was conducted in murine erythroleukemia cells and human umbilical cord derived erythroid progenitor (HUDEP) cells, modified by knocking out all the human α-globin genes. We identified ALS20α, a vector where α-globin is under control of the β-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20α produces exogenous α-globin at a level comparable to that produced by one endogenous α-globin gene. These results suggest that a relatively low VCN could result in dramatic therapeutic benefits. Transplantation of ALS20α transduced murine BM-KO results in correction of the disease phenotype in a dose-dependent manner. At VCN<1 we observe a delay in death proportional to the VCN value, while at VCN>1 we observe phenotypic normalization, including Hb, hepcidin and EPO levels.We tested ALS20α in CD34 cells isolated from four patients with both deletional and non- deletional HbH disease. We measured the change of β/α-globin mRNA ratio (β/αR) and protein level by HPLC in erythroblasts derived from these cultures. For the specimen with mutational HbH, the initial β/αR matches that of healthy controls, as the mutations do not eliminate the ability for the gene to produce aberrant mRNA transcripts, and decreased with increasing VCN. Erythroblasts with deletional HbH have a β/αR approximately 3x higher than normal cells, decreasing in a dose dependent manner with increasing VCN. HPLC detection of HbH (β4), a hallmark of HbH disease, is observed in hemolysis products from all non-transduced α−thal erythroblasts. A ~50% reduction of HbH is detected in the very same specimens upon integration of ALS20α (VCN between 1 and 2).In conclusion, we generated an adult mouse model of lethal α-thal and, in preliminary experiments, we rescue it with ALS20α. Furthermore, ALS20α successfully improves α-globin levels in patient cells. Further experiments are in progress to establish the consistency of our vector's expression in vivo, as well as to demonstrate its ability to transduce bona fide long-term HSCs. DisclosuresKattamis: Agios Pharmaceuticals: Consultancy; IONIS: Consultancy; VIFOR: Consultancy; CRISPR/Vertex: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding; Chiesi: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy. Rivella: Celgene Corporation: Consultancy; Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forma Theraputics: Consultancy; Incyte: Consultancy; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Genetic and Epigenetic Therapies for β-Thalassaemia by Altering the Expression of α-Globin Gene.

β-Thalassaemia is caused by over 300 mutations in and around the β-globin gene that lead to impaired synthesis of β-globin. The expression of α-globin continues normally, resulting in an excess of α-globin chains within red blood cells and their precursors. These unpaired α-globin chains form unstable α-hemichromes that trigger cascades of events to generate reactive oxygen species, leading to ineffective erythropoiesis and haemolysis in patients with β-thalassaemia. The clinical genetic data reported over several decades have demonstrated how the coinheritance of α-thalassaemia ameliorates the disease phenotype of β-thalassaemia. Thus, it is evident that down-regulation of the α-globin gene expression in patients with β-thalassaemia could ameliorate or even cure β-thalassaemia. Over the last few years, significant progress has been made in utilising this pathway to devise a cure for β-thalassaemia. Most research has been done to alter the epigenetic landscape of the α-globin locus or the well-characterised distant enhancers of α-globin. In vitro, pre-clinical studies on primary human erythroid cells have unveiled inhibition of histone lysine demethylation and histone deacetylation as potential targets to achieve selective downregulation of α-globin through epigenetic drug targeting. CRISPR based genome editing has been successfully used in vitro to mutate α-globin genes or enhancers of α-goblin to achieve clinically significant knockdowns of α-globin to the levels beneficial for patients with β-thalassaemia. This review summarises the current knowledge on the regulation of human α-globin genes and the clinical genetic data supporting the pathway of targeting α-globin as a treatment for β-thalassaemia. It also presents the progress of epigenetic drug and genome editing approaches currently in development to treat β-thalassaemia.

Identification of the transcription factor MAZ as a regulator of erythropoiesis

AbstractErythropoiesis requires a combination of ubiquitous and tissue-specific transcription factors (TFs). Here, through DNA affinity purification followed by mass spectrometry, we have identified the widely expressed protein MAZ (Myc-associated zinc finger) as a TF that binds to the promoter of the erythroid-specific human α-globin gene. Genome-wide mapping in primary human erythroid cells revealed that MAZ also occupies active promoters as well as GATA1-bound enhancer elements of key erythroid genes. Consistent with an important role during erythropoiesis, knockdown of MAZ reduces α-globin expression in K562 cells and impairs differentiation in primary human erythroid cells. Genetic variants in the MAZ locus are associated with changes in clinically important human erythroid traits. Taken together, these findings reveal the zinc-finger TF MAZ to be a previously unrecognized regulator of the erythroid differentiation program.

A gain-of-function single nucleotide variant creates a new promoter which acts as an orientation-dependent enhancer-blocker

Many single nucleotide variants (SNVs) associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. Here we show that a single base change in an otherwise unremarkable region of the human α-globin cluster creates an entirely new promoter and an associated unidirectional transcript. This SNV downregulates α-globin expression causing α-thalassaemia. Of note, the new promoter lying between the α-globin genes and their associated super-enhancer disrupts their interaction in an orientation-dependent manner. Together these observations show how both the order and orientation of the fundamental elements of the genome determine patterns of gene expression and support the concept that active genes may act to disrupt enhancer-promoter interactions in mammals as in Drosophila. Finally, these findings should prompt others to fully evaluate SNVs lying outside of known regulatory elements as causing changes in gene expression by creating new regulatory elements.

Mechanisms mediating suppression of globin gene transcription in Danio rerio nonerythroid cells

We studied the repression of adult and embryo-larval genes of the major globin gene locus in D. rerio fibroblasts. The results obtained suggest that at least some of the globin genes are repressed by Polycomb, similarly to human α-globin genes. Furthermore, within two α/β globin gene pairs, repression of α-type and β-type genes appears to be mediated by different mechanisms, as increasing the level of histone acetylation can activate transcription of only β-type genes.

A cytosine-rich splice regulatory determinant enforces functional processing of the human α-globin gene transcript

AbstractThe establishment of efficient and stable splicing patterns in terminally differentiated cells is critical to maintenance of specific functions throughout the lifespan of an organism. The human α-globin (hα-globin) gene contains 3 exons separated by 2 short introns. Naturally occurring α-thalassemia mutations that trigger aberrant splicing have revealed the presence of cryptic splice sites within the hα-globin gene transcript. How cognate (functional) splice sites are selectively used in lieu of these cryptic sites has remained unexplored. Here we demonstrate that the preferential selection of a cognate splice donor essential to functional splicing of the hα-globin transcript is dependent on the actions of an intronic cytosine (C)-rich splice regulatory determinant and its interacting polyC-binding proteins. Inactivation of this determinant by mutation of the C-rich element or by depletion of polyC-binding proteins triggers a dramatic shift in splice donor activity to an upstream, out-of-frame, cryptic donor. The essential role of the C-rich element in hα-globin gene expression is supported by its coevolution with the cryptic donor site in primate species. These data lead us to conclude that an intronic C-rich determinant enforces functional splicing of the hα-globin transcript, thus acting as an obligate determinant of hα-globin gene expression.

Hb H Disease Results from Compound Heterozygosity of – –SEA and –αMAL3.5 in a Chinese Family

The α+-thal deletion of 3.557 kb (NG_000006.1: g.32745_36301del, –αMAL3.5), involving the entire α2-globin gene, was identified in a Chinese family by multiplex ligation-dependent probe amplification (MLPA) followed by gap-polymerase chain reaction (gap-PCR) and sequencing. The proband, a compound heterozygote for this mutant gene and the Southeast Asian (– –SEA; NG_000006.1: g.26264_45564del19301) deletion, had a phenotype of Hb H disease [hemoglobin (Hb) 7.6 g/dL, mean corpuscular volume (MCV) 60.0 fL, Hb H (β4) 0.7%, Hb Bart’s (γ4) 2.4% and Hb A2 1.1%]; one of her sisters with same genotype showed a similar phenotype. Another two family members, who were carriers of this mutant gene, had a hematological phenotype of a silent α-thal. The 5' and 3' breakpoints of this deletion are located at the Y2 and Y1 boxes, respectively, therefore, it probably originated from an unequal crossover between these two homologous boxes. This mutation constitutes an additional heterogeneous defect causing α-thal in the Chinese population and would be valuable for elucidating the arrangement in the human α-globin gene cluster.

Two novel copy number variations involving the α-globin gene cluster on chromosome 16 cause thalassemia in two Chinese families.

Copy number variations (CNVs) can cause many genetic disorders and the structure analysis of unknown CNVs is important for clinical diagnosis. The human α-globin gene cluster lies close to the telomere of the short arm on chromosome 16. Copy number variations of this region produce excessive or insufficient α-globin chains which imbalances the β-globin chains, resulting in thalassemia. However, these CNVs usually cannot be precisely defined by traditional methods. Here, we designed a technique strategy and applied it to identify two CNVs involving the α-globin gene cluster causing thalassemia in two Chinese families. A novel 282kb duplication (αααα(282)) was identified in family A and a novel 235kb deletion (--(235)) in family B. Proband A is a coinheritance of β(CD41-42) and αααα(282) and showed severe β-thalassemia intermedia phenotype. Proband B is a compound heterozygote of --(235)/α(CS)α genotype and was diagnosed with hemoglobin H disease. The clinical phenotypic features of the CNVs carriers were described, together with a complete picture of molecular structure of these rearrangements. Two CNVs are novel rearrangements in α-globin clusters and the αααα(282) is the first to identify the exact insert position of a duplication region from the telomere on chromosome 16. In a conclusion, successful identification and characterization of these two novel CNVs not only demonstrates the precision and effectiveness of our strategy in analyzing the structure of unknown CNVs, but also extended the spectrum of thalassemia and provide new examples for studying genomic recombination.

Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). The level of sensitivity of a PTC-containing mRNA to NMD is multifactorial. We have previously shown that human β-globin mRNAs carrying PTCs in close proximity to the translation initiation AUG codon escape NMD. This was called the ‘AUG-proximity effect’. The present analysis of nonsense codons in the human α-globin mRNA illustrates that the determinants of the AUG-proximity effect are in fact quite complex, reflecting the ability of the ribosome to re-initiate translation 3′ to the PTC and the specific sequence and secondary structure of the translated ORF. These data support a model in which the time taken to translate the short ORF, impacted by distance, sequence, and structure, not only modulates translation re-initiation, but also impacts on the exact boundary of AUG-proximity protection from NMD.

α−thalassaemia: a genotype–phenotype correlation and management

Throughout the world, α−thalassaemia represents the most common monogenic disorder in man [1]. A large number of mutations (mainly deletions and a few nucleotide substitutions) of the human α-globin cluster have been described which interact to produce one of three broad phenotypes, α-thalassaemia heterozygote, Hb H disease and Hb Bart's hydrops fetalis [2]. In terms of public health, α-thalassaemia trait only causes a mild anaemia and Hb Bart's hydrops fetalis is usually fatal in late gestation [2-4]. In contrast, Hb H disease is a relatively common cause of thalassaemia in individuals of Southeast Asian, Middle Eastern and Mediterranean origins and is associated with a variety of clinically important complications [3, 5]. This review will focus on the molecular basis of α−thalassaemia in general and more specifically illustrate a recent development in molecular characterization of several, including novel, α−thalassaemia alleles found recently. The complexity of globin genotype–phenotype correlation in α−thalassaemia disease will be discussed with particular regard to patients with Hb H disease.

Phenotypic Characterization the Townes Sickle Mice

The HbSS-Townes mouse model, developed in Dr. Tim Townes laboratory, University of Alabama, Birmingham (and kindly provided by him to our laboratory) were created on a mixed genetic background in which the murine adult α-globin genes were replaced with the human α-globin gene (genotype: Hba hα/hα) and the murine adult β-globin genes were replaced with human sickle βS- and fetal Aγ-globin gene fragments linked together (genotype: Hbb hAγβS/hAγβS) (Wu LC et al. Blood 2006;108: 1183-1188). HbSS-Townes mice have anemia, a shortened RBC half-life of 2.5 days and a severe disease phenotype. Control HbAA-Townes mice were created by replacing the murine globin genes with human α-globin gene (genotype: Hba hα/hα) and linked human βA- and fetal Aγ-globins (genotype: Hbb hAγβA/hAγβA), while HbAS-Townes heterozygous mice were developed by breeding HbAA and HbSS mice. Many laboratories are utilizing these mice but complete phenotypic description of these three models has not been described including: hematology, kidney and liver function, inflammatory markers, haptoglobin and hemopexin levels, red cell half-lives, organ histopathology and vascular responses to vaso-occlusive stimuli. Table 1 summarizes our findings.There was a clear difference in histopathology between the HbSS and other groups (HbAA and HbAS). HbSS mice had hepatic necrosis, increased erythropoiesis and increased hemosiderin within the liver and some subtle lesions involving the glomeruli in the kidneys. Additional findings were a marked increase in size of spleen (7.0-7.6 x by % body weights) attributed to severe congestion and increased erythropoiesis. No lesions were observed in the lungs and other tissues. The tissues evaluated in the HbAS and HbAA groups were essentially normal. In contrast, HbSS mice had multifocal irregular areas of necrosis within the liver, with reactive leukocyte infiltrates (mainly neutrophils in the more acute lesions with a greater proportion of mononuclear cells (macrophages etc.) in more chronic lesions. MPO immunohistochemistry confirmed the presence of neutrophils in the liver. There was a substantial increase in iron (and hemosiderin) in the livers of HbSS mice, confirmed by a Prussian Perls stain and low, but detectable, levels of iron in the proximal convoluted tubules of the kidney. This is consistent with increased red cell turnover in the HbSS mice. Total iron mass in the markedly enlarged spleen is very high. There is a somewhat subtle glomerulopathy present in the kidney, with enlarged glomeruli with variable ectasia of vessels, and mesangial derangement. In conclusion the Townes mouse models provide a spectrum of severe hemolytic disease that in many ways mimic the human disease albeit imperfectly. TableTable 1HbAAHbASHbSSHb (g/dL)12.0 ± 0.610.6 ± 0.59.5 ± 1.4*Hematocrit (%)49.6 ± 2.345.0 ± 4.2*29.2 ± 0.9*#WBC (K/µL)10.8 ± 1.213.2 ± 4.038.2 ± 4.9*#Platelet Counts (K/uL)854 ± 78889 ± 1421004 ± 179Monocytes (%)8.0 ± 1.17.6 ± 1.77.4 ± 1.0Lymphocytes (%)60 ± 3.063.8 ± 5.667.5 ± 9.1Neutrophils (%)28.6 ± 5.926.1 ± 8.727.8 ± 2.2Reticulocytes (%)7.8 ± 1.78.6 ± 4.556.8 ± 2.6*#RBC Half-Life (days)15.710.62.4Expired CO (nmoles/h/g)0.92 ± 0.241.27 ± 0.236.33 ± 1.08*Serum Bilirubin (mg/dl)3.5 ± 0.94.3 ± 0.65.6 ± 0.4*Urine Creatinine (mg/dL)44.1 ± 3.845.9 ± 3.856.2 ± 7.0*Urine Osmolality (mOsm/kg H2O)2147 ± 761707 ± 2651361 ± 32*Serum Haptoglobin (µg/ml)39.3 ± 3.80.5 ± 0.2*2.3 ± 1.4*Serum Hemopexin (µg/ml)802 ± 266169 ± 51*124 ± 35*Serum SAP (µg/ml)20.9 ± 7.214.1 ± 12.086.7 ± 20.2*#Stasis at 1h in Response to Hb (%)10.0 ± 3.219.8 ± 2.7*30.0 ± 3.4*#Mortality at 24h in Response to Heme (%)00100*#Liver % of Body Weight4.95 ± 0.484.39 ± 0.356.22 ± 0.21*#Spleen % of Body Weight0.79 ± 0.140.87 ± 0.396.61 ± 0.75*#Kidney % of Body Weight0.65 ± 0.270.72 ± 0.190.73 ± 0.24Liver Necrosis Score0.0 ± 0.00.25 ± 0.502.38 ± 0.25*Liver Fe Score0.0 ± 0.00.25 ± 0.53.0 ± 0.0*Lung Fe Score0.0 ± 0.00.0 ± 0.0*1.0 ± 0.0*#Spleen Fe Score3.0 ± 0.03.0 ± 0.02.0 ± 0.0*#Kidney Fe Score0.0 ± 0.00.25 ± 0.51.75 ± 0.5*#Liver Gr1 Score1.0 ± 0.01.25 ± 0.52.25 ± 0.5*Lung Gr1 Score1.0 ± 0.01.0 ± 0.01.25 ± 0.5Spleen Gr1 Score1.5 ± 0.62.0 ± 0.02.0 ± 0.0Kidney Gr1 Score0.25 ± 0.50.25 ± 0.50.75 ± 0.5* p<0.05 vs HbAA; # p<0.05 vs HbAS DisclosuresNo relevant conflicts of interest to declare.

Differential regulation of the α-globin locus by Krüppel-like Factor 3 in erythroid and non-erythroid cells.

BackgroundKrüppel-like Factor 3 (KLF3) is a broadly expressed zinc-finger transcriptional repressor with diverse biological roles. During erythropoiesis, KLF3 acts as a feedback repressor of a set of genes that are activated by Krüppel-like Factor 1 (KLF1). Noting that KLF1 binds α-globin gene regulatory sequences during erythroid maturation, we sought to determine whether KLF3 also interacts with the α-globin locus to regulate transcription.ResultsWe found that expression of a human transgenic α-globin reporter gene is markedly up-regulated in fetal and adult erythroid cells of Klf3−/− mice. Inspection of the mouse and human α-globin promoters revealed a number of canonical KLF-binding sites, and indeed, KLF3 was shown to bind to these regions both in vitro and in vivo. Despite these observations, we did not detect an increase in endogenous murine α-globin expression in Klf3 −/− erythroid tissue. However, examination of murine embryonic fibroblasts lacking KLF3 revealed significant de-repression of α-globin gene expression. This suggests that KLF3 may contribute to the silencing of the α-globin locus in non-erythroid tissue. Moreover, ChIP-Seq analysis of murine fibroblasts demonstrated that across the locus, KLF3 does not occupy the promoter regions of the α-globin genes in these cells, but rather, binds to upstream, DNase hypersensitive regulatory regions.ConclusionsThese findings reveal that the occupancy profile of KLF3 at the α-globin locus differs in erythroid and non-erythroid cells. In erythroid cells, KLF3 primarily binds to the promoters of the adult α-globin genes, but appears dispensable for normal transcriptional regulation. In non-erythroid cells, KLF3 distinctly binds to the HS-12 and HS-26 elements and plays a non-redundant, albeit modest, role in the silencing of α-globin expression.

A Multiplex qPCR Gene Dosage Assay for Rapid Genotyping and Large-Scale Population Screening for Deletional α-Thalassemia

The predominant determinants of α-thalassemia are deletions in the human α-globin gene cluster. Arapid DNA-based assay is needed for mass screening in thalassemia-prevention programs. Herein, we established a novel quadruplex TaqMan qPCR gene dosage assay with two separate combination reactions. The assay directly determined the copy number of human α-globin genes based on relative quantitation of three target genes (HBA2, HBA1, and HBZ or HBPA1) versus a control gene (CREBBP). The assay showed good accuracy, with mean intra-assay and interassay variations of 3.31% ± 1.02% and 5.49% ± 0.32%, respectively. The assay was evaluated using 678 pretyped clinical DNA samples containing six α-thalassemia deletions in 13 genotypes and 186 normal samples previously screened by multiplex ligation-dependent probe amplification or gap PCR. As determined by the 2(-ΔΔCq) method, deleted gene dosage ratios were 0.46 to 0.60 in heterozygotes, 0.0 in homozygotes, and 0.97 to 1.07 in nondeleted samples. We found 99.3% concordance between the quantitative PCR and multiplex ligation-dependent probe amplification or gap-PCR results. Furthermore, routine screening for α-thalassemia deletions was performed on 3000 random samples in a blind analysis. Results for all 279 positives, which had different deletions, were fully coincident with results from standard methods. We also identified two novel deletions confirmed by multiplex ligation-dependent probe amplification. Assays using the novel method are simple and suitable for rapid genotyping and mass screening.

High-resolution analysis of cis -acting regulatory networks at the α-globin locus

We have combined the circular chromosome conformation capture protocol with high-throughput, genome-wide sequence analysis to characterize the cis-acting regulatory network at a single locus. In contrast to methods which identify large interacting regions (10-1000 kb), the 4C approach provides a comprehensive, high-resolution analysis of a specific locus with the aim of defining, in detail, the cis-regulatory elements controlling a single gene or gene cluster. Using the human α-globin locus as a model, we detected all known local and long-range interactions with this gene cluster. In addition, we identified two interactions with genes located 300 kb (NME4) and 625 kb (FAM173a) from the α-globin cluster.

Evidence of recent natural selection on the Southeast Asian deletion (--SEA) causing α-thalassemia in South China

BackgroundThe Southeast Asian deletion (--SEA) is the most commonly observed mutation among diverse α-thalassemia alleles in Southeast Asia and South China. It is generally argued that mutation --SEA, like other variants causing hemoglobin disorders, is associated with protection against malaria that is endemic in these regions. However, little evidence has been provided to support this claim.ResultsWe first examined the genetic imprint of recent positive selection on the --SEA allele and flanking sequences in the human α-globin cluster, covering a genomic region spanning ~410 kb, by genotyping 28 SNPs in a Chinese population consisting of 76 --SEA heterozygotes and 138 normal individuals. The pattern of linkage disequilibrium (LD) and the long-range haplotype test revealed a signature of positive selection. The network of inferred haplotypes suggested a single origin of the --SEA allele.ConclusionsThus, our data support the hypothesis that the --SEA allele has been subjected to recent balancing selection, triggered by malaria.

A quantitative assay to detect α-thalassemia deletions and triplications using multiplex nested real-time quantitative polymerase chain reaction

Increasing evidence indicates that copy number variants (CNVs) have great relevance to common human diseases. In α-thalassemia, clinical phenotypes are related to genotypes, specifically copy number changes in the human α-globin gene cluster. Assays are available for high-throughput screening of unknown CNVs genome-wide and also for targeted CNV genotyping at loci associated with genetic disorders. Here we describe a universal quantitative approach based on nested real-time quantitative polymerase chain reaction for accurate determination of copy numbers at multiple particular gene loci. We used the α-globin gene as a model system, obtaining the reproducibility and sensitivity to analyze different gene copies and testing 95 DNA samples with 16 different known genotypes. Our results showed that this approach has high sensitivity and low standard deviations for correctly genotyping DNA samples containing different copy numbers of the α1 and α2 globin genes. Our method is rapid, simple, and reliable, and it could be used to simultaneously screen for α-thalassemia deletions or triplications. Moreover, it has potential as a versatile technology for the rapid genotyping of known CNVs in a targeted region.

Nprl3 is required for normal development of the cardiovascular system

C16orf35 is a conserved and widely expressed gene lying adjacent to the human α-globin cluster in all vertebrate species. In-depth sequence analysis shows that C16orf35 (now called NPRL3) is an orthologue of the yeast gene Npr3 (nitrogen permease regulator 3) and, furthermore, is a paralogue of its protein partner Npr2. The yeast Npr2/3 dimeric protein complex senses amino acid starvation and appropriately adjusts cell metabolism via the TOR pathway. Here we have analysed a mouse model in which expression of Nprl3 has been abolished using homologous recombination. The predominant effect on RNA expression appears to involve genes that regulate protein synthesis and cell cycle, consistent with perturbation of the mTOR pathway. Embryos homozygous for this mutation die towards the end of gestation with a range of cardiovascular defects, including outflow tract abnormalities and ventriculoseptal defects consistent with previous observations, showing that perturbation of the mTOR pathway may affect development of the myocardium. NPRL3 is a candidate gene for harbouring mutations in individuals with developmental abnormalities of the cardiovascular system.

Kinetics of α-Globin Binding to α-Hemoglobin Stabilizing Protein (AHSP) Indicate Preferential Stabilization of Hemichrome Folding Intermediate

Human α-hemoglobin stabilizing protein (AHSP) is a conserved mammalian erythroid protein that facilitates the production of Hemoglobin A by stabilizing free α-globin. AHSP rapidly binds to ferrous α with association (k'(AHSP)) and dissociation (k(AHSP)) rate constants of ≈10 μm(-1) s(-1) and 0.2 s(-1), respectively, at pH 7.4 at 22 °C. A small slow phase was observed when AHSP binds to excess ferrous αCO. This slow phase appears to be due to cis to trans prolyl isomerization of the Asp(29)-Pro(30) peptide bond in wild-type AHSP because it was absent when αCO was mixed with P30A and P30W AHSP, which are fixed in the trans conformation. This slow phase was also absent when met(Fe(3+))-α reacted with wild-type AHSP, suggesting that met-α is capable of rapidly binding to either Pro(30) conformer. Both wild-type and Pro(30)-substituted AHSPs drive the formation of a met-α hemichrome conformation following binding to either met- or oxy(Fe(2+))-α. The dissociation rate of the met-α·AHSP complex (k(AHSP) ≈ 0.002 s(-1)) is ∼100-fold slower than that for ferrous α·AHSP complexes, resulting in a much higher affinity of AHSP for met-α. Thus, in vivo, AHSP acts as a molecular chaperone by rapidly binding and stabilizing met-α hemichrome folding intermediates. The low rate of met-α dissociation also allows AHSP to have a quality control function by kinetically trapping ferric α and preventing its incorporation into less stable mixed valence Hemoglobin A tetramers. Reduction of AHSP-bound met-α allows more rapid release to β subunits to form stable fully, reduced hemoglobin dimers and tetramers.

Development and Clinical Implementation of a Combination Deletion PCR and Multiplex Ligation-Dependent Probe Amplification Assay for Detecting Deletions Involving the Human α-Globin Gene Cluster

The α-thalassemias are a group of hereditary disorders caused by reduced synthesis of the α-chain of hemoglobin. We have developed and tested an α-thalassemia assay that uses both multiplex ligation-dependent probe amplification (MLPA) with Luminex-based detection and deletion PCR technologies. The MLPA assay consisted of 20 probes, 15 of which hybridized to the α-globin gene cluster and 5 that served as control probes. A PCR assay was developed to confirm the presence of heterozygous/homozygous 3.7-kb and 4.2-kb deletions. MLPA and PCR results were compared to Southern blot (SB) results from 758 and 133 specimens, respectively. Lastly, MLPA and PCR results were reviewed and summarized from 5386 clinically tested specimens. SB and MLPA results were concordant in 678/687 (99%) specimens. PCR detected all deletions detected by SB with no false positives. No deletions or duplications were identified in 2630 (49%) clinically tested specimens. Extra α-globin copies were identified in 76 patients. A deletion of one or two α-globin genes was identified in 1251 (23%) and 1349 (25%) specimens, respectively, including 15 different genotypes. A deletion of three (hemoglobin H) and four α-globin genes (Hb Bart's) was observed in 65 or 3 specimens, respectively. Six patients had a deletion within the α-globin regulatory region MCS-R2. Thus, MLPA plus deletion PCR identify multiple α-globin gene deletions/duplications in patients being tested for α-thalassemia.